mechanisms of development 136 (2015) 99–110

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Normal table of embryonic development in the four-toed salamander, Hemidactylium scutatum

C.A. Hurney a,*, S.K. Babcock a, D.R. Shook b, T.M. Pelletier a, S.D. Turner c, J. Maturo a, S. Cogbill a, M.C. Snow a, K. Kinch a a Department of Biology, MSC 7801, James Madison University, Harrisonburg, VA 22807 b Department of Biology, P.O. Box 400328, University of Virginia, Charlottesville, VA 22904 c Department of Public Health Sciences, University of Virginia School of Medicine, Charlottesville, Virginia 22908

ARTICLE INFO ABSTRACT

Article history: We present a complete staging table of normal development for the lungless salamander, Received 17 September 2014 Hemidactylium scutatum (Caudata: Plethodontidae). Terrestrial egg clutches from naturally Received in revised form ovipositing females were collected and maintained at 15 °C in the laboratory. Observations, 30 November 2014 photographs, and time-lapse movies of embryos were taken throughout the 45-day embry- Accepted 31 December 2014 onic period. The complete normal table of development for H. scutatum is divided into 28 Available online 21 January 2015 stages and extends previous analyses of H. scutatum embryonic development (Bishop, 1920; Humphrey, 1928). Early embryonic stage classifications through neurulation reflect criteria Keywords: described for Xenopus laevis, Ambystoma maculatum and other salamanders. Later embryonic Embryonic development stage assignments are based on unique features of H. scutatum embryos. Additionally, we Plethodontid provide morphological analysis of gastrulation and neurulation, as well as details on Normal table external aspects of eye, gill, limb, pigmentation, and tail development to support future re- Amphibian search related to phylogeny, comparative embryology, and molecular mechanisms of development. © 2014 Elsevier Ireland Ltd. All rights reserved.

a biphasic life mode. Adults are easy to identify due to a con- 1. Introduction striction at the base of the tail and a white venter with a distinct black spot pattern. H. scutatum exhibits a short-lived, aquatic Hemidactylium scutatum (Caudata: Plethodontidae) is a small, larval stage, which contrasts to the direct development mode four-toed salamander distributed throughout eastern North utilized by most members of the family Plethodontidae. Adult America. Characteristic of the family Plethodontidae, members females lay eggs during the late spring in nests of densely of the genus Hemidactylium lack lungs and possess nasolabial packed sphagnum moss patches alongside the edge of fresh grooves. As the sole species of the genus Hemidactylium and water ponds. Egg clutches develop in the terrestrial nests until the monotypic tribe Hemidactyliini (one of four tribes of the the gilled, aquatic larvae hatch and enter the pond for a short subfamily Hemidactyliinae), H. scutatum possesses four digits larval period resulting in metamorphosed juveniles that return on each hindlimb, mode V tongue feeding, and skulls with fully to terrestrial habitats during the summer. articulated frontal and parietal bones and no fontanelle (Wake, Broader understanding of embryonic morphological fea- 2012). Adults inhabit terrestrial, forested habitats with access tures and developmental patterns in H. scutatum may help to fishless ponds that serve as breeding locations to support resolve hypotheses regarding species diversity and the complex

* Corresponding author. James Madison University, USA. Tel.: +540 568 4830; fax: +540 568 4990. E-mail address: [email protected] (C.A. Hurney). http://dx.doi.org/10.1016/j.mod.2014.12.007 0925-4773/© 2014 Elsevier Ireland Ltd. All rights reserved. 100 mechanisms of development 136 (2015) 99–110 phylogenetic history of metamorphosis within the family 2.1. Major features of early development: stages 1–19 Plethodontidae (Bonett et al., 2005; Bruce, 2005; Chippindale (Tables 1 and 2; Figs. 1, 2, 3 and 4) and Wiens, 2005; Kerney et al., 2012). Short-lived, long-lived, and direct developing species are contained within the sub- 2.1.1. Cleavage stages (Stages 1–7; 0–27 hours after family Hemidactyliinae. Recent phylogenetic analyses indicate oviposition; Table 1; Fig. 1) that the monotypic tribe Hemidactyliini is the sister taxon to H. scutatum embryos have lightly pigmented animal poles the species-rich, direct-developing tribes Bolitoglossini and and no evidence of gray crescent pigmentation on the dorso- Batrachosepini (Vieites et al., 2011; Wake, 2012). Relations with lateral edge. Single-celled embryos average 2.4 mm in diameter members of the tribe Spelerpini, whose members exhibit long- (n = 7; range 2.3 mm−2.5 mm (Stage 1), which is consistent lived larval stages as well as aquatic paedomorphs, remain with previous reports from Humphrey (1928) and Bishop (1920). poorly resolved. As observed in most amphibians, cleavage in H. scutatum Despite the call for more studies on salamander embryon- embryos is asymmetrical and holoblastic. The first and second ic development, resolution of the debates regarding the cleavage planes in H. scutatum are meridional and orthogo- phylogenetic history of Plethodontidae is limited by the lack nal to one another. The first cleavage plane begins to form of comprehensive descriptions of embryonic morphogenesis approximately 8 hours after oviposition and division is com- (Bonett et al., 2005; Bruce, 2005; Chippindale and Wiens, 2005). plete within 12 hours (Stage 2). Before the first cleavage Most of the early reports of embryonic development in furrow is complete, a second furrow begins to form in the plethodontids are incomplete analyses based on field descrip- animal pole. The first two cleavages divide the embryo into tion or informal laboratory observations (Barden and Kezer, 1944; four cells by the end of Stage 3 (15 h). The third cleavage event Bishop, 1920, 1925, 1941; Bishop and Chrisp, 1933; Fowler, 1946; is equatorial and displaced toward the animal pole creating 4 Franz, 1967; Goin, 1951; Goodale, 1911; Hilton, 1909; Humphrey, micromeres in the animal hemisphere and 4 macromeres in 1928; Lynn and Dent, 1941; Miller, 1944; Rabb, 1956; Smith et al., the vegetal hemisphere observable externally (Stage 4, 18 h). 1968; Stebbins, 1949). More recent staging tables offer more in- After the 8-cell stage, cell division continues asymmetrically sights into embryonic features, but many are not comprehensive and synchronously, resulting in an increase of cell number or do not document the full embryonic period (Collazo and from 16 cells (Stage 5, 21 h) to 32 cells (Stage 6, 24 h) Keller, 2010; Collazo and Marks, 1994; Franssen et al., 2005; and ultimately to 64 cells (Stage 7, 27 h). Time-lapse movies Marks, 1995; Marks and Collazo, 1998). The embryonic staging at room temperature (22–23 °C) show that the interval table for the direct-developing Plethodon cinereus provides the between the onset of early cleavages decreases throughout most comprehensive analysis of plethodontid embryonic char- development. For example, 2 hours and 40 minutes elapse acters to date (Kerney, 2011). The staging table of embryonic between second and third cleavage, 2 hours and 10 minutes development for H. scutatum complements and extends pre- elapse between the third and fourth cleavage and 1 hour vious studies and provides a complete analysis of timing and and 40 minutes elapse between sixth and seventh cleavage. characterization of external development to extend knowl- Shorter intervals (1:35 or 1:30) occur for later cleavages. edge of salamander embryology, vertebrate development, and Thus developmental rates at room temperature are initially further enrich analyses of plethodontid life history evolution. only moderately faster than at 15 °C but tend toward twice as fast as the embryo progresses through cleavage stages. The overall size of the embryo increases slightly during cleav- age stages from 2.4 mm in diameter to 2.5 mm (n = 4). Similar asymmetric cleavage patterns are reported for other 2. Results plethodontids (Collazo, 1990; Collazo and Marks, 1994; Hilton, 1909; Kerney, 2011; Marks, 1995; Marks and Collazo, 1998), al- We define 28 embryonic stages for H. scutatum on the basis though field studies of two direct developing plethodontids, of external morphology. The total embryonic period from ovi- Aneides lugubris and Batrachoseps wrighti, report meroblastic position to hatching is approximately 45 days at 15 °C. Stages cleavage for early cleavage stages, followed by holoblastic cleav- 1 to 19 represent early development from cleavage through neu- age during later cleavage stages (Miller, 1944; Stebbins, 1949). rulation and are consistent with stage designations for events through neurulation defined for Ambystoma mexicanum 2.1.2. Blastula stages (Stages 8–9;1d16hto3d5hafter (Bordzilovskaya et al., 1989), Ambystoma maculatum (Harrison, oviposition; Table 1; Fig. 1) 1969), Desmognathus aeneus (Marks and Collazo, 1998), and H. scutatum embryos enter Stage 8 approximately 40 hours Xenopus laevis (Nieuwkoop and Faber, 1956). Stages 20 through after oviposition when the size of the apical aspect of the animal 28 document later developmental events such as tail, gill, and blastomeres decreases by a factor of 2 relative to the previ- limb formation culminating in hatching (Stage 28). After neu- ous stage. Asymmetric cell division continues, resulting in rulation, H. scutatum development deviates from described hundreds of animal blastomeres that remain smaller in size amphibian species and thus we define stages 20–28 based solely and greater in number than vegetal blastomeres. The next tran- on the appearance and change in external morphology. Land- sition occurs approximately 2 days and 9 hours after oviposition mark features of each stage are summarized (Tables 1–3) and when the animal hemisphere becomes smooth and lighter in illustrations of each stage are presented (Figs. 1–6), where each color (Stage 9). Manual bisection of Stage 8 and 9 H. scutatum illustration (or set of illustrations) depicts diagnostic external embryos confirmed the presence of a blastocoel roof two cell features that characterize a unique stage ofembryonic layers thick (data not shown). Embryos of the larval develop- development. ing plethodontid, Eurycea bislineata, have 2 cell layers (Goodale, mechanisms of development 136 (2015) 99–110 101

Table 1 – Descriptions of embryonic Stages 1–16 of Hemidactylium scutatum. Stage Approx. time Description since oviposition 1 0 hours One cell; the animal pole is heavily pigmented compared to the vegetal pole. The presence of the germinal vesicle is evident by an area of lighter pigmentation in the animal pole. The first cleavage furrow begins to form in the animal pole after 8 hours. 2 12 hours Two cells; the cleavage furrow expands meridionally through the animal pole to divide the embryo into two equal cells. The cleavage plane is not complete in the vegetal pole as the second furrow begins to form. 3 15 hours Four cells; a second meridional cleavage plane occurs perpendicular to the first and expands through the animal pole. Four blastomeres form when both divisions are complete. 4 18 hours Eight cells; the third cleavage is equatorial and displaced toward the animal pole, creating asymmetry with four micromeres in the animal pole and four macromeres in the vegetal pole 5 21 hours Sixteen cells; cell division continues synchronously, but asymmetrically in the animal and vegetal poles, creating 16 blastomeres. The animal pole remains darker than the vegetal pole. 6 24 hours Thirty-two cells; cell division continues asymmetrically, creating animal blastomeres that are approximately 1/2 the size of those in Stage 5. 7 27 hours Sixty-four cells; cell division continues asymmetrically, forming animal blastomeres that are approximately 1/2 the size of those in Stage 6. 8 1 day Early blastula; cell division becomes asynchronous and continues asymmetrically to form animal 16 hours blastomeres that are approximately 1/2 the size of those in Stage 7. 9 2days Late blastula; cell division continues and the surface of the animal cap is smooth. Cells are too 9 hours numerous to plausibly count and are individually pigmented. The animal pole is lighter than previous stages. 10 3 days Early gastrula; the first external sign of gastrulation appears as a dark blastopore lip on the 6 hours dorsovegetal surface of the embryo. The animal pole often appears wrinkled. 11 4 days Mid gastrula; as gastrulation continues, the blastopore lip elongates and becomes crescent shaped with the ends pointing ventrolaterally away from the future dorsal surface of the embryo. Involuting suprablastoporal cells are distinctly smaller than sub-blastoporal cells moving under the vegetal side of the blastopore lip. 12 5 days Late gastrula; blastopore lip shortens to form a deep horizontal slit that is darkly pigmented at the ends. 13 7 days Early neural plate; the blastopore lip elongates and becomes crescent shaped with the ends pointing anteriorly. The neural plate is slightly elevated from the dorsal surface and a shallow neural groove is present on anterior end of neural plate. The neural folds are not yet visible. 14 7 days Mid neural plate; the neural folds begin to emerge and elevate as the neural groove continues to 6 hours deepen along the anterior end of the plate. The blastopore lip flattens and decreases in length. 15 8 days Late neural plate; the neural folds continue to elevate and approach each other to form an “hourglass” shape. The neural groove is present along the length of the plate. 16 8 days Early neural fusion; the neural folds continue to elevate and approach with initial contact 3 hours occurring near the midbrain/hindbrain boundary of the forming neural tube. The blastopore is enclosed by the posterior end of the neural folds.

1911), while embryos of the larval developing Desmognathus zone tissue involutes around the dorsal and lateral blasto- fuscus have blastocoel roofs with only one cell layer (Hilton, pore lip while large amounts of tissue moves through the 1911). Ensatina eschscholtzii, a direct developing plethodontid, vegetal side of the blastopore. Unlike Ambystoma (Shook et al., also has embryos with one cell layer over the blastocoel (Collazo 2002), there is no involution of ventral marginal zone tissue and Keller, 2010). around a blastopore. As involution continues, the blastopore lip undergoes multiple shape changes that define Stages 11, 2.1.3. Gastrula stages (Stages 10–13;3d6hto7dafter 12, and 13 (4 d, 5 d, and 7 d, respectively; estimates from movies oviposition; Table 1; Fig. 2) at room temperature are 17 hours from Stages 11 to 12 and The first sign of gastrulation is evident approximately 3 days 20 hours from Stages 12 to 13). The blastopore lip elongates and 6 hours after oviposition when the blastopore lip appears to form a roughly third-circle crescent with ends that point lat- as a dark invagination on the dorso-vegetal side of embryos erally (Stage 11) that grows to a half-circle crescent which of H. scutatum (Stage 10). Time lapse movies show that a large persists 10 or 12 hours at room temperature, but keeps getting area of both dorsal marginal zone and dorsal vegetal endo- smaller. Rather than completing the blastopore ventrally, the dermal tissue apically contract to form the initial blastopore lip eventually shortens to form a horizontal slit with darkly lip, about 40–60 degrees in animal vegetal extent and 70–80 pigmented ends (Stage 12). The blastopore lip increases in length degrees in medio-lateral extent, forming a structure that is only again with ends that curve dorsally, around the posterior end a few degrees in animal vegetal extent, and about 30–45 degrees of the early neural plate (Stage 13). Movement of ventral tissue in medio-lateral extent. Endoderm around the vegetal pole also under the blastopore lip and involution around the dorsal lip undergoes a great deal of contraction prior to and during dorsal ends at or shortly after the transition from ventral-pointing blastopore lip formation. As gastrulation proceeds, marginal to dorsal pointing blastopore lip. Thus the flat blastopore lip 102 mechanisms of development 136 (2015) 99–110

Table 2 – Descriptions of embryonic Stages 17–21 of Hemidactylium scutatum. Stage Approx. time Description since oviposition 17 9 days Late neural fusion; the anterior folds are fused and the prospective brain regions are apparent. The folds continue to fuse posteriorly to form a “keyhole” shaped neural plate. 18 9 days Early neural tube; the neural folds are fused along the entire length of the embryo to form a 3 hours suture. Further extension and development of the cranial regions of the neural tube are apparent. A head fold begins to become apparent. 19 10 days Late neural tube; suture of neural tube is smooth along the length of the embryo. Neural development continues to progress as the most anterior regions of the developing brain continue to enlarge and the neural tissue as a whole extends. The head fold is more apparent and continues to separate the head from the ventral portion of the embryo. 20 11 days The branchial plate is divided into two mounds, the largest and most posterior is the gill mound. During this stage, the branchial plate divisions become more evident with the development of grooves between each arch. The blastopore is a small slit anterior to the tail bud. Anteriorly, the optic vesicles are prominent lateral to the closed neural tube and the head is undercut to the first branchial mound. The tail bud is evident as a swelling at the posterior end of the embryo. 21 14 days The forelimb bud (posterior) and the gill mound (anterior) are more prominent and spherical. The head is further undercut to the gill arches. The divisions of the branchial plate are more evident as the pharyngeal grooves clearly divide the mandibular, hyoid, and gill arches. The mandibular arch is prominent laterally and moves toward the ventral midline. The optic cup shows faint dorsal and lateral pigmentation and the lens placode invaginates to form the lens pit. The tail bud is rounded and undercut from the embryo. The embryo has a visible heartbeat and shows a motor response to mechanical stimuli.

stage appears to mark the approximate end of gastrulation. plate (Stage 13). During the next 6 hours, the neural folds emerge Time-lapse movies show that the dorsal curvature of the blas- as the neural groove continues to deepen along the length of topore is associated with the dorsal convergence of the neural the neural plate (Stage 14). As neurulation continues, the neural plate and the ectodermal tissue lateral to it. folds become more prominent and the blastopore lip flattens These observations are consistent with previous analysis again and decreases its width as the posterior neural folds close of H. scutatum embryos (Humphrey, 1928) but differ a great deal over it. During Stage 15 (8 d) the neural folds begin to form an from the pattern of blastopore closure observed in other anurans, “hourglass” shape. Initial contact and fusion of neural folds like X. laevis, in that there is no significant ventral lip, and shows occurs toward the anterior end, near the future midbrain/ substantial differences with the type of blastopore closure in hindbrain boundary (Stage 16, 8 d 3 h). The neural folds fuse other urodeles, such as Ambystoma, because such a large amount bi-directionally over the next 2 days: the anterior portion of of ventral tissue moves directly under the dorsal blastopore the neural tube forms by day 9 (Stage 17) while fusion of the lip, rather than involuting around a ventral lip (Keller and Shook, posterior region continues for another 3 hours, resulting in a 2004). In certain regards, gastrulation in H. scutatum re- completely sutured neural tube (Stage 18). Primary fusion of sembles gastrulation in reptiles (Bertocchini et al., 2013), as well the neural folds in the anterior region of the neural plate is as more primitive chordate gastrulation, in that there is no atypical of amphibians (Duellman and Trueb, 1986), though it ventral lip, which may be a primitive trait (discussed in Shook has been reported in one newt, Triturus pyrrhogaster (Anderson, and Keller, 2008). A complete yolk plug is formed in most larval 1943) and in the plethodontid salamander, E. bislineata (Goodale, and direct developers of the subfamily Plethodontinae with the 1911). Neurulation is complete by day 10 when the suture of exception of P. cinereus (Collazo, 1990; Dent, 1941; Marks, 1995; the neural tube is smooth (Stage 19). Cranial development begins Marks and Collazo, 1998). Among the Plethodontidae, only the during Stages 18 and 19 as the most anterior regions of the embryos of the larval developer, E. bislineata, have little to no developing brain begin to enlarge and extend. ventral blastopore and undergo similar changes in morphol- ogy as seen in H. scutatum during gastrulation (Goodale, 1911). 2.2. Major features of late development: Stages 20–28 In contrast, there are several exceptions to complete blasto- (Tables 2 and 3; Figs. 5, 6, and 7) pore formation in other families of urodeles as seen in some members of the genus, Ensatina, and the gigantic salamander, 2.2.1. Yolk reduction Megalobtrachus maximus (Collazo and Keller, 2010; Ichikawa, 1905). The ventral side of the H. scutatum embryo still contains much of the yolk volume 11 days after oviposition (Stage 20). 2.1.4. Neurula stages (Stages 13–19;7dto10dafter In contrast, direct developing salamanders such as A. lugubris oviposition; Tables 1 and 2; Figs. 3 and 4) and P. cinereus embryos develop upon a large ventral yolk mass Approximately 7 days after oviposition the neural plate of (Kerney, 2011; Miller, 1944; Piersol, 1910)orhaveyolken- H. scutatum embryos is slightly elevated with a shallow neural closed in abdominal folds (Marks and Collazo, 1998). groove and, as described above, the blastopore lip increases in Interestingly, Pseudotriton ruber, a larval developer, also exhib- length and curves around the posterior end of the early neural its a large ventral yolk mass (Bishop, 1925). The amount of yolk mechanisms of development 136 (2015) 99–110 103

Table 3 – Descriptions of embryonic Stages 22–28 of Hemidactylium scutatum. Stage Approx. time Description since oviposition 22 18 days The optic cup pigmentation is crescent shaped and becomes torus shaped with the choroid fissure still open at the end of the stage. The nasal pits are invaginated. The gill arch is tripartite and divides into three rami by the end of the stage. The cardiac prominence is evident. The amount of yolk is reduced. The length of the tail is approximately twice its dorsal-ventral height. 23 22 days The pigmentation of the optic cup forms a complete circle and the choroid fissure closes. The right and left mandibular arches meet in the ventral midline and fuse. The gular fold fuses in the ventral midline. The third gill rami elongate first; later in the stage, the second gill rami elongate to approximately the same length as the third gill rami. Melanophores, initially of greatest concentration at the dorsal branchial region, spread throughout the dorsal surface of the head and trunk to reach the gill rami, forelimbs, and base of the tail by the end of the stage. The tail flattens laterally. The dorsal fin extends anteriorly from the tip of the tail to the posterior half of the trunk. The ventral fin is evident from the cloaca to the tip of the tail. 24 25 days Optic cup pigmentation intensifies. The third gill rami are the first to branch, followed by the second typically a day later. The median notch of the gular fold indents and the edges of the fold become more distinct, forming a flap. Melanophores increase in concentration along the length of the head, trunk and tail. Bilaterally staggered spots begin to form along the dorsum of the anterior trunk, suggestive of the future larval pigmentation pattern. At the beginning of the stage, the distal end of the forelimb is polygonal. The hindlimb bud is present anterior to the cloaca and has melanophores on its dorsum. The dorsal fin extends anteriorly, spanning nearly 3/4 the length of the trunk. Both the dorsal and ventral fins increase in height. 25 28 days The ventral cranial tissues become translucent between the mandibular cartilage and the gular fold. All three gill rami are branched; the second and third rami are branched more than the first. The larval pigmentation pattern is evident as the melanophore concentration increases, xanthophores appear along the dorsum and forelimbs, and the dorsal spot pattern extends throughout the trunk and into the tail. A few melanophores appear on the pericardium. The forelimb axis begins to change shape as wrist and elbow joints differentiate. The second digit extends beyond the first and third digit buds. By the end of the stage, the distal end of the hindlimb bud is paddle shaped. The vitelline vein is prominent ventrally and lateral vascularization increases as the amount of yolk decreases. 26 33 days The forelimb has three distinct digits. A dark horizontal stripe forms, passing through the eye and nasal pits along the lateral aspect of the head. Green-reflecting iridophores appear in the eye. The median notch of the gular fold further indents. Pericardial pigmentation increases. Toward the end of the stage, the most anterior section of the gut curves into s-shapes. Xanthophores have migrated to the tail and are diffusely scattered in the ventral and dorsal fins. 27 37 days The gular fold is biconvex and deepens. Pericardial pigmentation intensifies laterally. The forelimb has four digits; the second digit is greatest in length, followed by the third, first, and fourth. The elbow joint can bend to 90 degrees. Curving continues in the anterior part of the gut. The hindlimb buds are polygon shape with digit buds indicating the future first, second, and third toes. The knee joint has begun to differentiate. 28 45 days The eyes are laterally placed and lidless, and have gold-reflecting iridophores. A few dark yellow xanthophores are diffusely scattered on the head, gills, forelimbs, and spots along the dorsum. The gills are elongate and highly branched. The second and third digits of the forelimb are equal in length. The venter is translucent from the cranium to the tail. The gut forms shaped patterns along the entire length. The hindlimb has three distinct digits and occasionally the fourth digit bud has begun to extend.

in H. scutatum embryos decreases during the next two stages rounded tail bud that extends posteriorly (Stage 21, 14 d). Ap- while lateral ventral blood vessels become prominent by Stage proximately 4 days later the tail elongates to become twice as 22. A more prominent blood vessel appears along the ventral long as its dorso-ventral height (Stage 22, 18 d). The tail begins side of the embryo and the amount of yolk continues to de- to flatten laterally and the tip becomes more pointed. The dorsal crease during Stages 25 to 27. At hatching, a small amount of fin extends anteriorly from the tail tip to the posterior half of yolk persists posterior to the curved gut (Fig. 5). Hatchlings of the trunk while the ventral fin extends from the tip toward the larval developers, Gyrinophilus porphyriticus, E. bislineata and cloaca (Stage 23, 22 d). During Stage 24 (25 d) the dorsal fin D. fuscus also exhibit reduced yolk (Collazo, 1990, 1996; Goin, extends further anteriorly to span nearly 3/4 the length of the 1951). trunk and the ventral fin extends to the posterior border of the cloaca. Both fins continue to increase in height as the tail 2.2.2. Tail elongation lengthens (Stages 25 through 28). The resulting broad, keel- The tail bud forms as a swelling just dorsal to the small slit- shaped tail is typical of pond-type larvae at hatching (Duellman shaped blastopore about 1 day after the completion of and Trueb, 1986; Goin, 1951; Rabb, 1956), rather than stream neurulation (Stage 20, 11 d). Elongation results in a bluntly dwellers (Petranka, 1998). 104 mechanisms of development 136 (2015) 99–110

Fig. 1 – Sketches of cleavage Stages 1–9 of Hemidactylium scutatum highlighting dorsal views. 1 mm scale bar included for size reference.

2.2.3. Eye development The gill arch enlarges over the next 4 days and forms three gill Optic vesicles are prominent in the developing head rami (Stage 22, 18 d).The third gill ramus elongates during Stage of H. scutatum embryos approximately 1 day after the 23 (22 d) followed by elongation of the second then first gill ramus. completion of neurulation (Stage 20, 11 d). Faint pigmenta- The third and second gill rami branch extensively during Stage tion appears on the developing optic cup during the next 3 days 24 (25 d), while the first gill ramus branches during Stage 25 (28 d). and the lens placode invaginates to form the lens pit (Stage Melanophores are present on all rami. Gill rami and branches 21, 14 d). By the end of Stage 22 (18 d), optic cup pigmenta- continue to elongate until hatching. A similar sequence of gill tion, which is initially crescent-shaped, intensifies around the rami formation, elongation, and branching occurs in Desmognathus edges to become torus-shaped with an open choroid fissure. ochrophaeus, Desmognathus quadramaculatus, G. porphyriticus, The choroid fissure closes to produce optic pigmentation that P. cinereus, B. wrighti,A. lugubris, and E. eschscholtzii (Collazo, 1990; fully encircles the lens pit (Stage 23, 22 d). Optic pigmenta- Collazo and Marks, 1994; Dent, 1941; Marks, 1995; Marks and tion continues to darken as the cornea forms (Stage 24, 25 d). Collazo, 1998; Miller, 1944). In contrast, the second rami branch Melanophores begin to form a dark horizontal stripe (Stage 26, first during gill development in D. aeneus, while the first rami 33 d) that becomes continuous across the lateral aspect of the branch first in Batrachoseps attenuatus embryos (Collazo, 1990; head and snout by Stage 28 (45 d). Green and gold iridiophores Marks and Collazo, 1998). The extent of embryonic branching appear concurrently with the formation of the horizontal stripe. in the gills varies throughout the family Plethodontidae. Larval Like other larval developers, H. scutatum hatchlings have lat- developers (D. quadramaculatus, G. porphyriticus) and direct de- erally placed, lidless eyes (Bishop, 1925; Collazo and Marks, 1994; velopers (D. aeneus,andP. cinereus) have long rami that are highly Marks, 1995), rather than the forward-oriented eyes of direct branched similar to the rami described for H. scutatum (Collazo developers (Collazo, 1990; Dent, 1941; Kerney, 2011; Marks and and Marks, 1994; Kerney, 2011; Marks and Collazo, 1998). Collazo, 1998). D. ochrophaeus,A. lugubris, and E. eschscholtzii have short gills that do not branch or are not highly branched (Collazo, 1990; Marks, 2.2.4. Gill development and branching 1995; Miller, 1944). The branchial plate is visible in H. scutatum embryos ap- At hatching, the pond-type larvae of H. scutatum have highly proximately 1 day after the completion of neurulation (Stage 20, branched, long gills similar to Eurycea quadridigitata and 11 d). Pharyngeal grooves deepen during Stage 21 (14 d) and clearly Stereochilus marginatus (Petranka, 1998). Stream-type larvae typ- delineate the gill arch from the mandibular and hyoid arches. ically have shorter gills that are not extensively branched, as mechanisms of development 136 (2015) 99–110 105

Fig. 2 – Sketches of gastrula Stages 10–13 of Hemidactylium scutatum highlighting dorsal views. 1 mm scale bar included for size reference. reported for G. porphyriticus, D. ochrophaeus, and P. ruber (Bishop, 2.2.5. Limb formation 1925; Collazo and Marks, 1994; Marks, 1995). By contrast, the The forelimb bud begins forming posterior to the bran- gills of direct developers (B. wrighti, B. attenuatus, E. eschscholtzii, chial bud approximately 1 day after the completion of Bolitoglossa subpalmata, Bolitoglossa compacta, A. lugubris, P. cinereus, neurulation (Stage 20, 11 d) and is prominent by Stage 21 (14 d). and D. aeneus) are typically resorbed at the time of hatching Initially, the distal end of the forelimb bud is initially round or shortly thereafter (Collazo, 1990; Collazo and Marks, 1994; (Stage 21, 14 d), then paddle-shaped, and ultimately polygo- Dent, 1941; Hanken, 1979; Marks and Collazo, 1998; Miller, 1944; nal in shape just prior to the appearance of digits (Stage 24, Stebbins, 1949). 25 d). The forelimb undergoes multiple changes during Stage

Fig. 3 – Sketches of neurula Stages 14–19 of Hemidactylium scutatum highlighting dorsal views. 1 mm scale bar included for size reference. 106 mechanisms of development 136 (2015) 99–110

Fig. 4 – Sketches of embryonic Stages 17–21 of Hemidactylium scutatum highlighting select anterior, lateral, ventral and posterior views. 1 mm scale bar included for size reference.

25 (28 d), including the presence of melanophores and forelimb is well developed with the respective digit lengths, xanthophores on the dorsal surface, the differentiation of wrist 2 = 3 > 1 > 4 (Stage 28, 45 d). and elbow joints, and the appearance of digits. At Stage 26 (33 d), The hindlimb bud appears at Stage 24 (25 d; digit 2 is notably greater in length than digit 3 and the newly approximately 11 days after the appearance of the forming digit 1. Four digits are present by Stage 27 (37 d) with forelimb bud) with melanophores present on the dorsal the respective lengths being digit 2 > 3 > 1 > 4. Forelimbs are surface. The distal end of the hindlimb bud is initially mobile and bend 90° at the elbow. As hatching approaches, the round and becomes paddle-shaped by the end of Stage

Fig. 5 – Sketches of embryonic Stages 22–24 of Hemidactylium scutatum highlighting select dorsal, lateral and ventral views. 2 mm scale bar included for size reference. mechanisms of development 136 (2015) 99–110 107

Fig. 6 – Sketches of embryonic Stages 25–28 of Hemidactylium scutatum highlighting select dorsal and lateral views. 2 mm scale bar included for size reference.

25 (28 d) and polygonal in shape just prior to the appearance some larval developers have almost synchronous appear- of digits (Stage 27, 37 d). A differentiated knee and three ance and development of the forelimbs and hindlimbs, as digits, occasionally four, are present at hatching (Stage 28, reported in D. aeneus, D. quadramaculatus, D. ochrophaeus, 45 d). E. eschscholtzii, and B. attenuatus (Collazo, 1990; Marks, 1995; Forelimbs typically develop before the hindlimbs in most Marks and Collazo, 1998). The adult complement of forelimb of the described plethodontid larval developers, including digits is well formed at the time of hatching in H. scutatum, while H. scutatum, G. porphyriticus, P. ruber, Desmognathus monticola, and the hindlimbs rarely display fully developed digits. Similar pat- E. bislineata (Bishop, 1925; Collazo, 1990; Marks and Collazo, 1998) terns have been reported for the larval developer G. porphyriticus and one direct developer, B. subpalmata. Direct developers and (Collazo and Marks, 1994). Direct developers (P. cinereus, D. aeneus)

Fig. 7 – Sketches of embryonic Stages 25–28 of Hemidactylium scutatum highlighting select ventral and lateral views. 2 mm scale bar included for size reference. 108 mechanisms of development 136 (2015) 99–110

and some larval developers (A. lugubris, D. fuscus) have the adult at 15 °C on moistened filter paper; those without membranes complement of digits on both forelimbs and hindlimbs at hatch- were submerged in artificial pond water (Wyngaard ing (Dent, 1941; Goin, 1951; Hilton, 1909; Marks and Collazo, and Chinnappa, 1982). All images were collected at room 1998; Miller, 1944; Piersol, 1910). temperature and specimens were returned to 15 °C immediately. 2.2.6. Pigmentation changes Melanophores first appear dorsal to the branchial region 3.2. Monitoring embryonic development and migrate throughout the dorsal surface of the head and Observations and photographs of 32 individual, unanes- trunk to reach the gill rami, forelimbs, and base of tail (Stage thetized embryos were taken at intervals of 12 or 24 hour using 23, 22 d). The yolk gives a yellowish color to the venter. Mela- a Leica Wild M8 microscope with a Hamamatsu CCD camera nophore pigmentation intensifies along the dorso-lateral length (2002, 2004, 2005) or a Zeiss Stemi SV6 microscope with an Insight of the embryo and a series of staggered bilateral spots (absence camera (2006). Photographs of clutch-mates were taken at less of melanophores) appear on the anterior dorsum of the trunk frequent intervals. Images were time-stamped such that in- (Stage 24, 25 d). A few days later the dorsal spot pattern extends tervals between observable changes in morphology were throughout the trunk and tail while xanthophores appear along documented. All linear and area measurements were collect- the dorsum and forelimbs; anterior ventral tissues become ed from images cited above using Oncor 1.7 (2002, 2004, 2005) translucent between the mandible and gular fold (Stage 25, and Spot v.4 (2006) image analysis software. Representative image 28 d). Melanophores appear in the pericardium (Stage 25) and sets for each stage can be found in the Dryad Digital Repository. pigmentation intensifies during Stages 26 and 27 (33–37 d). Time-lapse sequences (1 frame every 5 minutes) during cleav- Xanthophores migrate into the tail and are diffusely scat- age, gastrulation, and neurulation were compiled from about tered in dorsal and ventral fins (Stage 26, 33 d). As the yolk 50 embryos maintained at 21–23 °C. Images were captured using reduces, the heart, blood vessels, stomach, and other organs a Zeiss SV6 microscope with a Dage MTI CCD-72 camera via can be seen through the skin (Stages 26 to 28). A few dark NIH image 1.6 or Scion image capture software or using an yellow xanthophores are present on the head, gills, fore- Olympus IX70 inverted microscope with a Hamamatsu CCD- limbs, trunk and tail (Stage 28, 45 d). The larval pigmentation 4742 camera via Metamorph software. Representative movies pattern is established by hatching (Stage 28, 45) and consists can be found in the Dryad Digital Repository. of 6 to 8 pairs of staggered yellowish spots on a mottled dark Six embryos were fixed in Smith’s fixative, placed in 6% dorsum with a translucent venter and darkly pigmented agarose gel, and bisected to confirm the presence of a blasto- pericardium. coel. The surfaces of two embryos were stained with a Development of the species-specific larval pigmentation crystallized piece of Nile Blue using fine forceps to establish pattern in H. scutatum is similar to the process observed in the pattern of neural tube fusion. embryos of larval developing plethodontids, including G. porphyriticus, Eurycea longicauda, Eurycea lucifuga, E. quadridigitata, Desmognathus auriculatus, D. quadramaculatus, 3.3. Preparation of developmental series D. ochrophaeus, and P. ruber (Bishop, 1925; Collazo and Marks, Total developmental time for H. scutatum (reared at 15 °C) 1994; Franz, 1967; Goin, 1951; Hutchison, 1958; Marks, 1995). was estimated by combining data from multiple embryos from In contrast, hatchlings of direct developers (P. cinereus, several clutches with overlapping developmental stages. We B. attenuatus, E. eschscholtzii, D. aeneus, and B. wrighti) display pig- were able to confirm time of oviposition for one embryo ovi- mentation patterns that reflect the adult pigmentation (Collazo, posited by an adult female maintained in the lab. Thirteen of 1990; Dent, 1941; Marks and Collazo, 1998; Piersol, 1910; the 32 individuals photographed at regular intervals were docu- Stebbins, 1949). mented for 30 or more consecutive days; individuals undergoing key changes in morphology were photographed more fre- quently. Clutch mates of multiple individuals were maintained 3. Experimental procedures in jelly coats and egg membranes and reared to natural hatching. Our goal in dividing the embryonic period of H. scutatum into 3.1. Collection and maintenance of Hemidactylium discrete developmental stages was to facilitate meaningful com- scutatum embryos parison with existing amphibian staging tables and to generate a useful tool for experimentation in which each stage re- Portions of terrestrial egg clutches (n = 13) from naturally flects readily observable changes in external morphology.Thus, ovipositing females were collected from the George Washing- each stage represents a period of time during which diagnos- ton National Forest in Rockingham (2002, 2004) and Botetourt tic features undergo prominent changes. Hand-drawn (2005, 2006) counties of Virginia. One naturally ovipositing female illustrations highlight stage-specific features and were gener- from Rockingham County was maintained on moistened ated to scale using multiple images. Principal diagnostic features filter paper and moss in the lab for less than one week are those typical of amphibian staging tables (Bordzilovskaya (2004). Field collected embryos ranged from day 1 to day et al., 1989; Harrison, 1969; Marks and Collazo, 1998; Nieuwkoop 18 of the estimated 45-day embryonic period. Jelly coats and Faber, 1956) and include cell number, formation of a blas- and egg membranes were removed from some embryos tocoel, shape of blastopore, formation of the neural tube, yolk manually using fine forceps; representative clutch-mates reduction, and aspects of the development of the tail, eyes, gills, were intact until hatching. All embryos were maintained limbs, and pigmentation. mechanisms of development 136 (2015) 99–110 109

For over a century developmental biologists have been docu- 4. Discussion menting stages of amphibian embryonic development. Seminal contributions include the normal table of development of This study provides the first comprehensive description of Xenoups laevis by Nieuwkoop and Faber (1956) and contribu- the embryonic development of the plethodontid salamander, tions on the development of Ambystoma maculatum embryos Hemidactylium scutatum. Embryos of the four-toed salaman- (Harrison, 1969) and Ambystoma mexicanum (Bordzilovskaya et al., der develop into free-swimming larvae over a 45-day period 1989). Although not often viewed as results, these tools rep- represented by 28 distinct stages. With regard to early embry- resent some of the most cited papers in developmental biology onic development (Stages 1–19), our results confirm previous (Hopwood, 2007). More importantly, these tools provide a foun- work on H. scutatum (Bishop, 1920; Humphrey, 1928) yet are de- dation for research into complex developmental phenomena, tailed enough to facilitate comparisons with amphibian model plasticity in life history evolution, and phylogenetic analyses. organisms (e.g. Xenopus and Ambystoma) in terms of timing, se- Further studies of this nature will aid interspecific compari- quence, and pattern variations. Later developmental stages sons within and outside the family Plethodontidae and may (Stages 20–28) reveal diagnostic features that may prove useful reveal underlying developmental patterns or features among for additional research related to the life history evolution in larval and direct developers. the family Plethodontidae and comparative embryological studies with other salamanders. Embryos of H. scutatum exhibit asymmetric, holoblastic cleav- Acknowledgements age pattern resulting in a blastula with a two-cell layer thick blastocoel roof. Gastrulation commences with the formation We want to acknowledge the undergraduate and graduate of a blastopore lip that undergoes numerous shape changes students who worked tirelessly on this project. We are grate- while marginal zone and vegetal tissue involutes and moves ful to our colleagues who supported this research by providing through the blastopore. The neural plate forms an hour-glass a stimulating academic work environment. Funding for this shape with primary fusion of the neural folds occurring in the research came from a grant from the Jeffress Memorial Trust anterior region of the embryo and closes bi-directionally forming (J-807) to Carol Hurney, and a NIH merit award (R37HD025594) the neural tube. 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